FLT3 Mutations Determine the Clinical Outcome in Children with De Novo Acute Myelogenous Leukemia (AML) and Normal Karyotype: Pediatric Oncology Group (POG) Study # 9421.

Abstract

The karyotype of the leukemia cell at diagnosis is of prognostic importance. The presence of t(8;21), inv(16) and t(15;17) are currently used to make therapeutic decisions. Additionally, specific mutations such as those involving the FLT3 gene are of prognostic importance as they indicate patients likely to relapse early or fail initial induction therapy. Approximately 20% of children with AML have normal karyotypes at diagnosis and no identifiable chromosomal abnormality using standard methods of analysis. In this subgroup there is an increased incidence of FLT3 mutations (internal tandem duplications or point mutations). We observed that patients with normal karyotypes who were enrolled in the Pediatric Oncology Group (POG) study #9421 had two significantly different clinical outcomes that were associated with the expression of FLT3 mutations. We hypothesized that gene expression profiles would identify genes that cooperate with FLT3 mutations in conferring poor clinical outcome.Bone marrow and/or blood samples from an initial 103 of 622 eligible patients registered on the POG study #9421 were studied. Cytogenetic testing was carried out by clinical laboratories at institutions where AML was diagnosed; all cytogenetic reports underwent centralized review. Samples were analyzed on a 43,760-element spotted array (containing 41,751 unique genes and ESTs) from the Stanford University Microarray Core Facility. Of the 103 diagnostic samples, 20 had normal karyotypes. The FLT3-mutation status was determined by RT-PCR analysis on RNA samples (exon 14 for ITD's and exon 20 for point mutations). Twelve patients expressed FLT3-mutations and 8 expressed wild-type FLT3 (FLT3-WT). Both groups had a mean WBC of 103x109/L at diagnosis. Probability of event free survival (EFS) was 75% for the FLT3-WT subgroup and 9% for the FLT3-mutant subgroup (P=0.0006). We used Prediction Analysis for Microarrays (PAM) to find the minimum number of genes that could identify samples with FLT3 mutations. PAM identified a 7-gene cluster that differentiated normal cytogenetic cases by clinical outcome. A cluster of 5 overexpressed genes (i.e., HIST1H2AC, HIST1H2AL, HIST1H2BD, HIST1H2BG, and HIST2H2BE) and 2 underexpressed genes (GASP and IVNS1ABP) were associated with poor outcome.In summary, children with AML, normal karyotype, and absence of FLT3 mutations have a good outcome. In contrast, when these same patients express a FLT3 mutation their outcome is poor despite intensive therapy. This poor outcome is associated with increased expression of several histone genes, which may be downstream targets of the FLT3 signal transduction pathway. We will corroborate these findings in a larger set of POG 9421 diagnostic samples with normal karyotypes.